Monoclonal antibody to a human T cell antigen and methods of preparing same

ABSTRACT

Hybrid cell line for production of monoclonal antibody to an antigen found on essentially all normal human T cells and on approximately 95% of normal human thymocytes. The hybrid is formed by fusing splenocytes from immunized CAF 1  mice with P3X63Ag8U1 myeloma cells. Diagnostic and therapeutic uses of the monoclonal antibody are also disclosed.

FIELD OF THE INVENTION

This invention relates generally to new hybrid cell lines and morespecifically to hybrid cell lines for production of monoclonal antibodyto an antigen found on essentially all normal human periphal T cells andon approximately 95% of normal human thymocytes, to the antibody soproduced, and to therapeutic and diagnostic methods and compositionsemploying this antibody.

DESCRIPTION OF THE PRIOR ART

The fusion of mouse myeloma cells to spleen cells from immunized mice byKohler and Milstein in 1975 [Nature 256, 495-497 (1975)] demonstratedfor the first time that it was possible to obtain a continuous cell linemaking homogeneous (so-called "monoclonal") antibody. Since this seminalwork, much effort has been directed to the production of various hybridcells (called "hybridomas") and to the use of the antibody made by thesehybridomas for various scientific investigations. See, for example,Current Topics in Microbiology and Immunology, Volume 81--"LymphocyteHybridomas", F. Melchers, M. Potter, and N. Warner, Editors,Springer-Verlag, 1978, and references contained therein; C. J.Barnstable, et al., Cell, 14, 9-20 (May, 1978); P. Parham and W. F.Bodmer, Nature 276, 397-399 (November, 1978); Handbook of ExperimentalImmunology, Third Edition, Volume 2, D. M. Wier, Editor, Blackwell,1978, Chapter 25; and Chemical and Engineering News, Jan. 1, 1979,15-17. These references simultaneously indicate the rewards andcomplications of attempting to produce monoclonal antibody fromhybridomas. While the general technique is well understood conceptually,there are many difficulties met and variations required for eachspecific case. In fact, there is no assurance, prior to attempting toprepare a given hybridoma, that the desired hybridoma will be obtained,that it will produce antibody if obtained, or that the antibody soproduced will have the desired specificity. The degree of success isinfluenced principally by the type of antigen employed and the selectiontechnique used for isolating the desired hybridoma.

The attempted production of monoclonal antibody to human lymphocytecell-surface antigens has been reported only in a few instances. See,for example, Current Topics in Microbiology and Immunology, ibid, 66-69and 164-169. The antigens used in these reported experiments werecultured human lymphoblastoid leukemia and human chronic lymphocyticleukemia cell lines. Many hybridomas obtained appeared to produceantibody to various antigens on all human cells. None of the hybridomasproduced antibody against a predefined class of human lymphocytes.

More recently, the present applicants and others have authored articlesdisclosing the preparation and testing of hybridomas making antibody tocertain T-cell antigens. See, for example, Reinherz, E. L., et al., J.Immunol. 123, 1312-1317 (1979); Reinherz, E. L., et al., Proc. Natl.Acad. Sci., 76, 4061-4065 (1979); and Kung, P. C., et al., Science, 206,347-349 (1979).

It should be understood that there are two principal classes oflymphocytes involved in the immune system of humans and animals. Thefirst of these (the thymus-derived cell or T cell) is differentiated inthe thymus from haemopoietic stem cells. While within the thymus, thedifferentiating cells are termed "thymocytes." The mature T cells emergefrom the thymus and circulate between the tissues, lymphatics, and thebloodstream. These T cells form a large proportion of the pool ofrecirculating small lymphocytes. They have immunological specificity andare directly involved in cell-mediated immune responses (such as graftrejection) as effector cells. Although T cells do not secrete humoralantibodies, they are sometimes required for the secretion of theseantibodies by the second class of lymphocytes discussed below. Sometypes of T cells play a regulating function in other aspects of theimmune system. The mechanism of this process of cell cooperation is notyet completely understood.

The second class of lymphocytes (the bone marrow-derived cells or Bcells) are those which secrete antibody. They also develop fromhaemopoietic stem cells, but their differentiation is not determined bythe thymus. In birds, they are differentiated in an organ analogous tothe thymus, called the Bursa of Fabricius. In mammals, however, noequivalent organ has been discovered, and it is thought that these Bcells differentiate within the bone marrow.

It is now recognized that T cells are divided into at least severalsubtypes, termed "helper", "suppressor", and "killer" T cells, whichhave the function of (respectively) promoting a reaction, suppressing areaction, or killing (lysing) foreign cells. These subclasses are wellunderstood for murine systems, but they have only recently beendescribed for human systems. See, for example, R. L. Evans, et al.,Journal of Experimental Medicine, Volume 145, 221-232, 1977; and L.Chess and S. F. Schlossman - "Functional Analysis of Distinct HumanT-Cell Subsets Bearing Unique Differentiation Antigens", in"Contemporary Topics in Immunobiology", O. Stutman, Editor, PlenumPress, 1977, Volume 7, 363-379.

The ability to identify or suppress classes or subclasses of T cells isimportant for diagnosis or treatment of various immunoregulatorydisorders or conditions.

For example, certain leukemias and lymphomas have differing prognosisdepending on whether they are of B cell or T cell origin. Thus,evaluation of the disease prognosis depends upon distinguishing betweenthese two classes of lymphocytes. See, for example, A. C. Aisenberg andJ. C. Long, The American Journal of Medicine, 58:300 (March, 1975); D.Belpomme, et al., in "Immunological Diagnosis of Leukemias andLymphomas", S. Thierfelder, et al., eds, Springer, Heidelberg, 1977,33-45; and D. Belpomme, et al., British Journal of Haematology, 1978,38, 85.

Certain disease states (e.g., juvenile rheumatoid arthritis,malignancies, and agammaglobulinemia) are associated with an imbalanceof T cell subclasses. It has been suggested that autoimmune diseasesgenerally are associated with an excess of "helper" T cells or adeficiency of certain "suppressor" T cells, while agammaglobulinemia isassociated with an excess of certain "suppressor" T cells or adeficiency of "helper" T cells. Malignancies generally are associatedwith an excess of "suppressor" T cells.

In certain leukemias, excess T cells are produced in an arrested stageof development. Diagnosis may thus depend on the ability to detect thisimbalance or excess and to determine which developmental stage is inexcess. See, for example, J. Kersey, et al., "Surface Markers DefineHuman Lymphoid Malignancies with Differing Prognoses" in Haematology andBlood Transfusion, Volume 20, Springer-Verlag, 1977, 17-24, andreferences contained therein; and E. L. Reinherz, et al., J. Clin.Invest., 64, 392-397 (1979).

Acquired agammaglobulinemia, a disease state in which no immune globulinis produced, comprises at least two distinct types. In type I thefailure to produce immune globulin is due to an excess of suppressor Tcells, while in type II it is due to a lack of helper T cells. In bothtypes, there appears to be no defect or lack in the patients' B cells,the lymphocytes which are responsible for the actual secretion of theantibody; however, these B cells are being either suppressed or "nothelped", resulting in greatly decreased or absent immune globulinproduction. The type of acquired agammaglobulinemia may thus bedetermined by testing for an excess of suppressor T cells or an absenceof helper T cells.

On the therapeutic side, there is some suggestion, as yet not definitelyproven, that administration of antibodies against the subtype of T cellin excess may have therapeutic benefit in autoimmune disease ormalignancies. For example, a helper T cell cancer (certain cutaneous Tcell lymphomas and certain T cell acute lymphoblastic leukemias) may betreated by an antibody to a helper T cell antigen. Treatment ofautoimmune disease caused by an excess of helper cells may also beaccomplished in the same fashion. Treatment of diseases (e.g.,malignancies or type I acquired agammaglobulinemia) due to an excess ofsuppressor T cells may be treated by administration of an antibody to asuppressor T cell antigen.

Antisera against the entire class of human T cells (so-called antihumanthymocyte globulin or ATG) has been reported useful therapeutically inpatients receiving organ transplants. Since the cell-mediated immuneresponse (the mechanism whereby transplants are rejected) depends upon Tcells, administration of antibody to T cells prevents or retards thisrejection process. See, for example, Cosimi, et al., "RandomizedClinical Trial of ATG in Cadaver Renal Allgraft Recipients: Importanceof T Cell Monitoring", Surgery 40:155-163 (1976) and referencescontained therein; and Wechter, et al., "Manufacture of AntithymocyteGlobulin for Clinical Trials", Transplantation, 28 (4), 303-307 (1979).

The identification and suppression of human T cell classes andsubclasses has previously been accomplished by the use of spontaneousautoantibodies or selective antisera for human T cells obtained byimmunizing animals with human T cells, bleeding the animals to obtainserum, and adsorbing the antiserum with (for example) autologous but notallogeneic B cells to remove antibodies with unwanted reactivities. Thepreparation of these antisera is extremely difficult, particularly inthe adsorption and purification steps. Even the adsorbed and purifiedantisera contain many impurities in addition to the desired antibody,for several reasons. First, the serum contains millions of antibodymolecules even before the T cell immunization. Second, the immunizationcauses production of antibodies against a variety of antigens found onall human T cells injected. There is no selective production of antibodyagainst a single antigen. Third, the titer of specific antibody obtainedby such methods is usually quite low, (e.g., inactive at dilutionsgreater than 1:100) and the ratio of specific to non-specific antibodyis less than 1/10⁶.

See, for example, the Chess and Schlossman article referred to above (atpages 365 and following) and the Chemical and Engineering News articlereferred to above, where the deficiencies of prior art antisera and theadvantages of monoclonal antibody are described.

SUMMARY OF INVENTION

There has now been discovered a novel hybridoma (designated OKT11) whichis capable of producing novel monoclonal antibody against an antigenfound on essentially all normal human peripheral T cells and onapproximately 95% of normal human thymocytes, but not on normal B cellsor null cells.

The antibody so produced is monospecific for a single determinant onessentially all normal human peripheral T cells and contains essentiallyno other anti-human immune globulin, in contrast to prior art antisera(which are inherently contaminated with antibody reactive to numeroushuman antigens) and to prior art monoclonal antibodies (which are notmonospecific for a human T cell and thymocyte antigen). Moreover, thishybridoma can be cultured to produce antibody without the necessity ofimmunizing and killing animals, followed by the tedious adsorption andpurification steps necessary to obtain even the impure antisera of theprior art.

It is accordingly one object of this invention to provide hybridomaswhich produce antibodies against an antigen found on essentially all ofnormal human peripheral T cells.

It is a further aspect of the present invention to provide methods forpreparing these hybridomas.

A further object of the invention is to provide essentially homogeneousantibody against an antigen found on essentially all normal humanperipheral T cells.

A still further object is to provide methods for treatment or diagnosisof disease employing this antibody.

Other objects and advantages of the invention will become apparent fromthe examination of the present disclosure.

In satisfaction of the foregoing objects and advantages, there isprovided by this invention a novel hybridoma producing novel antibody toan antigen found on essentially all normal human peripheral T cells andon approximately 95% of normal human thymocytes (but not on normal humanB cells or null cells), the antibody itself, and diagnostic andtherapeutic methods employing the antibody. The hybridoma was preparedgenerally following the method of Milstein and Kohler. Followingimmunization of mice with leukemic cells from a human with T-cell acutelymphoblastic leukemia, (T-ALL), the spleen cells of the immunized micewere fused with cells from a mouse myeloma line and the resultanthybridomas screened for those with supernatants containing antibodywhich gave selective binding to normal E rosette positive human T cellsand/or E⁻ human cells. The desired hybridomas were subsequently clonedand characterized. As a result, a hybridoma was obtained which producesantibody (designated OKT11) against an antigen on essentially all normalhuman peripheral T cells. Not only does this antibody react withessentially all normal human peripheral T cells, but it also reacts withabout 95% of normal human thymocytes but does not react with normalhuman B cells or null cells.

In view of the difficulties indicated in the prior art and the lack ofsuccess reported using malignant cell lines as the antigen, it wassurprising that the present method provided the desired hybridoma. Itshould be emphasized that the unpredictable nature of hybrid cellpreparation does not allow one to extrapolate from one antigen or cellsystem to another. In fact, the present applicants have discovered thatthe use of a T cell malignant cell line or purified antigens separatedfrom the cell surface as the antigen were generally unsuccessful.

Both the subject hybridoma and the antibody produced thereby areidentified herein by the designation "OKT11", the particular materialreferred to being apparent from the context. The subject hybridoma wasdeposited on Dec. 13, 1979 at the American Type Culture Collection,12301 Parklawn Dr., Rockville, Md. 20852, and was given the ATCCaccession number CRL 8027.

The preparation and characterization of the hybridoma and the resultantantibody will be better understood by reference to the followingdescription and Examples.

DETAILED DESCRIPTION OF THE INVENTION

The method of preparing the hybridoma generally comprises the followingsteps:

A. Immunizing mice with leukemic cells from a human with T-ALL. While ithas been found that female CAF₁ mice are preferred, it is contemplatedthat other mouse strains could be used. The immunization schedule andthymocyte concentration should be such as to produce useful quantitiesof suitably primed splenocytes. Three immunizations at fourteen dayintervals with 2×10⁷ cells/mouse/injection in 0.2 ml phosphate bufferedsaline has been found to be effective.

B. Removing the spleens from the immunized mice and making a spleensuspension in an appropriate medium. About one ml of medium per spleenis sufficient. These experimental techniques are well-known.

C. Fusing the suspended spleen cells with mouse myeloma cells from asuitable cell line by the use of a suitable fusion promoter. Thepreferred ratio is about 5 spleen cells per myeloma cell. A total volumeof about 0.5-1.0 ml of fusion medium is appropriate for about 10⁸splenocytes. Many mouse myeloma cell lines are known and available,generally from members of the academic community or various depositbanks, such as the Salk Institute Cell Distribution Center, La Jolla,CA. The cell line used should preferably be of the so-called "drugresistant" type, so that unfused myeloma cells will not survive in aselective medium, while hybrids will survive. The most common class is8-azaguanine resistant cell lines, which lack the enzyme hypoxanthineguanine phophoribosyl transferase and hence will not be supported by HAT(hypoxanthine, aminopterin, and thymidine) medium. It is also generallypreferred that the myeloma cell line used be of the so-called"non-secreting" type, in that it does not itself produce any antibody,although secreting types may be used. In certain cases, however,secreting myeloma lines may be preferred. While the preferred fusionpromoter is polyethylene glycol having an average molecular weight fromabout 1000 to about 4000 (commercially available as PEG 1000, etc.),other fusion promoters known in the art may be employed.

D. Diluting and culturing in separate containers, the mixture of unfusedspleen cells, unfused myeloma cells, and fused cells in a selectivemedium which will not support the unfused myeloma cells for a timesufficient to allow death of the unfused cells (about one week). Thedilution may be a type of limiting one, in which the volume of diluentis statistically calculated to isolate a certain number of cells (e.g.,1-4) in each separate container (e.g., each well of a microtiter plate).The medium is one (e.g., HAT medium) which will not support the drugresistant (e.g., 8-azaguanine resistant) unfused myeloma cell line.Hence, these myeloma cells perish. Since the unfused spleen cells arenon-malignant, they have only a finite number of generations. Thus,after a certain period of time (about one week) these unfused spleencells fail to reproduce. The fused cells, on the other hand, continue toreproduce because they possess the malignant quality of the myelomaparent and the ability to survive in the selective medium of the spleencell parent.

E. Evaluating the supernatant in each container (well) containing ahybridoma for the presence of antibody to E rosette positive purifiedhuman T cells or thymocytes.

F. Selecting (e.g., by limiting dilution) and cloning hybridomasproducing the desired antibody.

Once the desired hybridoma has been selected and cloned, the resultantantibody may be produced in one of two ways. The purest monoclonalantibody is produced by in vitro culturing of the desired hybridoma in asuitable medium for a suitable length of time, followed by recovery ofthe desired antibody from the supernatant. The suitable medium andsuitable length of culturing time are known or are readily determined.This in vitro technique produces essentially monospecific monoclonalantibody, essentially free from other specific antihuman immuneglobulin. There is a small amount of other immune globulin present sincethe medium contains xenogeneic serum (e.g., fetal calf serum). However,this in vitro method may not produce a sufficient quantity orconcentration of antibody for some purposes, since the concentration ofmonoclonal antibody is only about 50 μg/ml.

To produce a much greater concentration of slightly less pure monoclonalantibody, the desired hybridoma may be injected into mice, preferablysyngenic or semi-syngenic mice. The hybridoma will cause formation ofantibody-producing tumors after a suitable incubation time, which willresult in a high concentration of the desired antibody (about 5-20mg/ml) in the bloodstream and peritoneal exudate (ascites) of the hostmouse. Although these host mice also have normal antibodies in theirblood and ascites, the concentration of these normal antibodies is onlyabout 5% of the monoclonal antibody concentration. Moreover, since thesenormal antibodies are not antihuman in their specificity, the monoclonalantibody obtained from the harvested ascites or from the serum isessentially free of any contaminating antihuman immune globulin. Thismonoclonal antibody is high titer (active at dilutions of 1:50,000 orhigher) and high ratio of specific to non-specific immune globulin(about 1/20). Immune globulin produced incorporating the light myelomachains are non-specific, "nonsense" peptides which merely dilute themonoclonal antibody without detracting from its specificity.

EXAMPLE I Production of Monoclonal Antibodies

A. Immunization and Somatic Cell Hybridization

Female CAF₁ mice (Jackson Laboratories; 6-8 weeks old) were immunizedintraperitoneally with 2×10⁷ human leukemic T-ALL cells in 0.2 ml ofphosphate buffered saline at 14-day intervals. Four days after the thirdimmunization, spleens were removed from the mice, and a single cellsuspension was made by pressing the tissue through a stainless steelmesh.

Cell fusion was carried out according to the procedure developed byKohler and Milstein. 1×10⁸ splenocytes were fused in 0.5 ml of a fusionmedium comprising 35% polyethylene glycol (PEG 1000) and 5%dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, NY) with2×10⁷ P3X63Ag8U1 myeloma cells supplied by Dr. M. Scharff, AlbertEinstein College of Medicine, Bronx, NY. These myeloma cells secreteIgG₁ light chains.

B. Selection and Growth of Hybridoma

After cell fusion, cells were cultured in HAT medium (hypoxanthine,aminopterin, and thymidine) at 37° C. with 5% CO₂ in a humid atmosphere.Several weeks later, 40 to 100 l of supernatant from cultures containinghybridomas were added to a pellet of 10⁶ peripheral lymphocytesseparated into E rosette positive (E⁺) and E rosette negative (E⁻)populations, which were prepared from blood of healthy human donors asdescribed by Mendes (J. Immunol. 111:860, 1973). Detection of mousehybridoma antibodies binding to these cells was determined by indirectimmunofluorescence. Cells incubated with culture supernatants werestained with a fluorescinated goat-anti-mouse IgG (G/M FITC) (MeloyLaboratories, Springfield, VA; F/p=2.5) and the fluorescentantibody-coated cells were subsequently analyzed on the CytofluorografFC200/4800A (Ortho Instruments, Westwood, MA) as described in ExampleIII. Hybridoma cultures containing antibodies reacting specifically withE⁺ lymphocytes (T cells) and/or thymocytes were selected and clonedtwice by limiting dilution methods in the presence of feeder cells.Subsequently, the clones were transferred intraperitoneally by injecting1×10.sup. 7 cells of a given clone (0.2 ml volume) into CAF₁ mice primedwith 2,6,10,14-tetramethylpentadecane, sold by Aldrich Chemical Companyunder the name Pristine. The malignant ascites from these mice were thenused to characterize lymphocytes as described below in Example II. Thesubject hybrid antibody OKT11 was demonstrated by standard techniques tobe of IgG₁ subclass.

EXAMPLE II Characterization of OKT11 Reactivity

A. Isolation of Lymphocyte Populations

Human peripheral blood mononuclear cells were isolated from healthyvolunteer donors (ages 15-40) by Ficoll-Hypaque density gradientcentrifugation (Pharmacia Fine Chemicals, Piscataway, NJ) following thetechnique of Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97): 77,1968. Unfractionated mononuclear cells were separated into surface Ig⁺(B) and Ig⁻ (T plus Null) populations by Sephadex G-200 anti-F(ab')₂column chromatography as previously described by Chess, et al., J.Immunol. 113:1113 (1974). T cells were recovered by E rosetting the Ig⁻population with 5% sheep erythrocytes (microbiological Associates,Bethesda, MD). The rosetted mixture was layered over Ficoll-Hypaque andthe recovered E⁺ pellet treated with 0.155M NH₄ Cl (10 ml per 10⁸cells). The T cell population so obtained was <2% EAC rosette positiveand >95% E rosette positive as determined by standard methods. Inaddition, the non-rosetting Ig⁻ (Null cell) population was harvestedfrom the Ficoll interface. This latter population was <5% E⁺ and >2%sIg⁺. The surface Ig⁺ (B) population was obtained from the SephadexG-200 column following elution with normal human gamma globulin aspreviously described. This population was >95% surface Ig⁺ and <5% E⁺.

B. Isolation of Thymocytes

Normal human thymus gland was obtained from patients aged two months to14 years undergoing corrective cardiac surgery. Freshly obtainedportions of the thymus gland were immediately placed in 5% fetal calfserum in medium 199 (Gibco), finely minced with forceps and scissors,and subsequently made into single cell suspensions by being pressedthrough wire mesh. The cells were next layered over Ficoll-Hypaque andspun and washed as previously described in section A above. Thethymocytes so obtained were >95% viable and ≧90% E rosette positive.

EXAMPLE III Cytofluorographic Analysis and Cell Separation

Cytofluorographic analysis of monoclonal antibodies with all cellpopulations was performed by indirect immunofluorescence withfluorescein-conjugated goat anti-mouse IgG (G/M FITC) (MeloyLaboratories) utilizing a Cytofluorograf FC200/4800A (OrthoInstruments). In brief, 1×10⁶ cells were treated with 0.15 ml OKT5 at a1:500 dilution, incubated at 4° C. for 30 minutes, and washed twice. Thecells were then reacted with 0.15 ml of a 1:40 dilution G/M FITC at 4°C. for 30 minutes, centrifuged, and washed three times. Cells were thenanalyzed on the Cytofluorograf, and the intensity of fluorescence percell was recorded on a pulse height analyzer. A similar pattern ofreactivity was seen at a dilution of 1:10,000, but further dilutioncaused loss of reactivity. Background staining was obtained bysubstituting a 0.15 ml aliquot of 1:500 ascites from a CAF₁ mouseintraperitoneally injected with a non-producing hybrid clone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fluorescence pattern obtained on the Cytofluorografafter reacting normal human thymocytes and E⁺ and E⁻ peripheral cellswith OKT11 and other monoclonal antibodies at a 1:500 dilution and G/MFITC. Background fluorescence staining was obtained by incubating eachpopulation with a 1:500 dilution of ascitic fluid from a mouse injectedwith a non-producing clone.

The production of the hybridoma and the production and characterizationof the resulting monoclonal antibody were conducted as described in theabove Examples. Although large quantities of the subject antibody wereprepared by injecting the subject hybridoma intraperitoneally into miceand harvesting the malignant ascites, it is clearly contemplated thatthe hybridoma could be cultured in vitro by techniques well-known in theart and the antibody removed from the supernatant.

Table 1 shows the reactivity of OKT1, OKT3-6, OKT8-11 with various humanlymphoid cell populations. This pattern of reactivity is one test bywhich the subject antibody OKT11may be detected and distinguished fromother antibodies.

FIG. 1 shows a representative fluorescense pattern obtained on theCytofluorograf after reacting normal human thymocyte suspensions, and E⁻and E+ peripheral cells with a 1:500 dilution of OKT11, OKT10, OKT8, andG/M FITC. In contrast to OKT1 and OKT3 T cell antigens (which increasewhen thymocytes mature to peripheral T cells), that of OKT11 antigenconcomitantly decreases. The pattern of reactivity in FIG. 1 is anothertest by which the subject antibody OKT11 may be detected anddistinguished from other antibodies.

Table 2 shows the antigen phenotypes of humsan T lineage lymphocytes,using OKT11 and other monoclonal antibodies. This phenotype patternprovides a still further way to detect OKT11 antibody and distinguish itfrom other antibodies.

Table 3 shows the relationship between levels of peripheral T cells andT cell subsets and various disease states. These relationships may beused for diagnositc purposes (e.g., to detect acute infectiousmononucleosis) by analyzing the blood sample of an individual suspectedof having one of these disease states to determine the levels of T cellsand T cell subsets. These relationships may also be used for therapeuticpurposes where the cause of the disease state is an elevated level of aT cell subset (e.g., Type I acquired agammaglobulinemia). Fortherapeutic use, administration of the appropriate monoclonal antibodyto a patient with an elevated T cell subset level will decrease oreliminate the excess. The relationships shown in Table 3 are a furtherway in which OKT11 antibody may be detected and distinguished from otherantibodies.

Other monoclonal antibody producing hybridomas prepared by the presentapplicants (designated OKT1, OKT3, OKT4, and OKT5) are described andclaimed in the following U.S. patent applications: Ser. Nos. 22,132,filed Mar. 20, 1979; 33,639, filed Apr. 26, 1979; 33,669, filed Apr. 26,1979; and 76,642, filed Sept. 18, 1979; and 82-515, filed Oct. 9, 1979.Still other monoclonal antibody producing hybridomas prepared by thepresent applicants (designated OKT6, OKT8, OKT9, and OKT10) aredescribed and claimed in U.S. patent applications filed on Dec. 4, 1979,and entitled: Hybrid Cell Line for Producing Monoclonal Antibody to aHuman Thymocyte Antigen, Antibody, and Methods; Hybrid Cell Line ForProducing Complement-Fixing Monoclonal Antibody to Human Suppressor TCells, Antibody, and Methods; Hybrid Cell Line For Producing MonoclonalAntibody to Human Early Thymocyte Antigen, Antibody, and Methods; andHybrid Cell Line For Producing Monoclonal Antibody to a HumanProthymocyte Antigen, Antibody, and Methods. A further hybridomaprepared by the present applicants (designated OKM1) is described andclaim in a U.S. patent application filed on even date herewith andentitled: Hybrid Cell Line for Producing Monoclonal Antibody to a HumanMonocyte Antigen, Antibody, and Methods.

These applications are incorporated herein by reference.

According to the present invention there are provided a hybridomacapable of producing antibody against an antigen found on essentiallyall peripheral T cells and approximately 95% of normal human thymocytes,a method for producing this hybridoma, monoclonal antibody against anantigen found on essentially all normal human peripheral T cells and onapproximately 95% of normal human thymocytes, methods for plroducing theantibody, and methods and compositions for treatment or diagnosis ofdisease or identification of T cell subclasses employing this antibody.

                                      TABLE 1                                     __________________________________________________________________________    Cellular and Tissue Distribution of OKT Antigens                                          Peripheral                                                        Monoclonal  Blood (15)*     Bone                                              Antibody                                                                             Ig Type                                                                            E.sup.+                                                                            E.sup.-                                                                            Spleen (4)                                                                          Marrow (6)                                                                          Thymus (15)                                 __________________________________________________________________________    OKT1   IgG.sub.1                                                                            100**                                                                            0    29 ± 9                                                                           <5    18 ± 8                                   OKT3   IgG.sub.2a                                                                         100  0    31 ± 8                                                                           <5    21 ± 8                                   OKT4   IgG.sub.2b                                                                         64 ± 4                                                                          0    16 ± 3                                                                           <5     75 ± 10                                 OKT5   IgG.sub.1                                                                          25 ± 4                                                                          0    15 ± 4                                                                           <5    77 ± 8                                   OKT6   IgG.sub.1                                                                          0    0    0     <5    68 ± 8                                   OKT8   IgG.sub.2a                                                                         34 ± 3                                                                          0    17 ± 4                                                                           <5    81 ± 7                                   OKT9   IgG.sub.1                                                                          0    0    0     <5     4 ± 3                                   OKT10  IgG.sub.1                                                                           5 ± 3                                                                          13 ± 4                                                                          10 ± 5                                                                           16 ± 4                                                                           96 ±  4                                  OKT11.sup.++                                                                         IgG.sub.1                                                                          100  0    NT.sup.+                                                                            NT    95 ± 3                                   __________________________________________________________________________     *number of samples examined                                                   **mean ± IS.D.                                                             .sup.+ not determined                                                         .sup.++ five peripheral blood and thymus samples were examined           

                                      TABLE 2                                     __________________________________________________________________________    Antigen Phenotypes of Human T Lineage Lymphocytes                                              OKT1                                                                              OKT3                                                                              OKT4                                                                              OKT5                                                                              OKT6                                                                              OKT8                                                                              OKT9                                                                              OKT10                                                                             OKT11                        __________________________________________________________________________          Prothymocyte                                                                             -   -   -   -   -   -   -   +   -                                  Early Thymocyte                                                                          -(W)                                                                              -(W)                                                                              +   -   -   -   ±                                                                              +   +                            Thymus                                                                              Common Thymocyte                                                                         -(W)                                                                              -(W)                                                                              +   +   +   +   ±                                                                              +   +                                  Mature hymocyte                                                                          +   +   +   -   -   -   -   +   +                                             +   +   -   +   -   +   -   +   +                            Peripheral                                                                          Inducer (Helper)                                                                         +   +   +   -   -   -   -   -   +                            T Cells                                                                             Cytotoxic/Suppressor                                                                     +   +   -   +   -   +   -   -   +                            __________________________________________________________________________     (W) = very weak immunofluorescence                                       

                                      TABLE 3                                     __________________________________________________________________________    PERIPHERAL MONONUCLEAR CELL LEVELS IN DISEASE STATES                                      Mononuclear Cell Levels                                           Disease State                                                                             OKT3.sup.+                                                                         OKT4.sup.+                                                                          OKT5                                                                              OKT8                                                                              OKT6                                                                              OKT11                                                                             OKM1                                   __________________________________________________________________________    Primary Biliary                                                                           N    +     -   -   -   ++  -                                      Cirrhosis (2)                                                                 Multiple Sclerosis                                                                        -    N     -   -   -   -   +                                      (advanced disease) (8)                                                        Myasthenia Gravis                                                                         O    O     O   O   O   -   +                                      (early untreated) (3)                                                         Acute Graft vs. Host (3)                                                                  O to -                                                                             -     O   +   +   +   -                                      Acquired Agamma                                                               globulinemia                                                                   Type I                +                                                       Type II         O                                                            Hyper IgE (4)                                                                             -    N     O to -                                                                            O to -                                                                            -   +   N                                      Acute Infectious Mono-                                                                    +    O to --                                                                             ++  ++  O   ++  -                                      nucleosis (4)*                                                                Hodgkins Disease                                                               Stages I & II                                                                            N    N     N   N   O   O   N                                       Stages III & IV                                                                          --   N     N   N   O   O   ++                                     Psoriasis (3/5)                                                                           N    + to ++                                                                             N   N   O   N   N                                      __________________________________________________________________________     N = within normal limits                                                      O = absent                                                                    + = above normal                                                              ++ = greatly above normal                                                     - = below normal                                                              -- = greatly below normal                                                     *these levels return to normal about one week prior to the disappearance      of clinical symptoms                                                          The numbers in parentheses indicate the number of patients evaluated.    

Although only a single hybridoma producing a single monoclonal antibodyagainst a human thymocyte antigen is described, it is contemplated thatthe present invention encompasses all monoclonal antibodies exhibitingthe characteristics described herein. It was determined that the subjectantibody OKT11 belongs to the subclass IgG₁, which is one of foursubclasses of murine IgG. These subclasses of immune globulin G differfrom one another in the so-called "fixed" regions, although an antibodyto a specific antigen will have a so-called "variable" region which isfunctionally identical regardless of which subclass of immune globulin Git belongs to. That is, a monoclonal antibody exhibiting thecharacteristic described herein may be of subclass IgG₁, IgG₂ a, IgG₂ b,or IgG₃, or of classes IgM, IgA, or other known Ig classes. Thedifferences among these classes or subclasses will not affect theselectivity of the reaction pattern of the antibody, but may affect thefurther reaction of the antibody with other materials, such as (forexample) complement or anti-mouse antibodies. Although the subjectantibody is specifically IgG₁, it is contemplated that antibodies havingthe patterns of reactivity illustrated herein are included within thesubject invention regardless of the immune globulin class or subclass towhich they belong.

Further included within the subject invention are methods for preparingthe monoclonal antibodies described above employing the hybridomatechnique illustrated herein. Although only one example of a hybridomais given herein, it is contemplated that one skilled in the art couldfollow the immunization, fusion, and selection methods provided hereinand obtain other hybridomas capable of producing antibodies having thereactivity characteristics described herein. Since the individualhybridoma produced from a known mouse myeloma cell line and spleen cellsfrom a known species of mouse cannot be further identified except byreference to the antibody produced by the hybridoma, it is contemplatedthat all hybridomas producing antibody having the reactivitycharacteristics described above are included within the subjectinvention, as are methods for making this antibody employing thehybridoma.

Further aspects of the invention are methods of treatment or diagnosisof disease employing the monoclonal antibody OKT11 or any othermonoclonal antibody exhibiting the pattern of reactivity providedherein. The subject antibody may be employed to diagnose disease statesas shown in Table 3. These techniques may be employed using OKT11antibody alone or in combination with other antibodies (e.g., OKT3 -OKT10). Patterns of reactivity with a panel of antibodies to T cells andT cell subsets will allow more precise detection of certain diseasestates then is possible using prior diagnostic methods.

Treatment of undesired states manifesting themselves as an excess ofOKT11⁺ cells may be accomplished by administration of a therapeuticallyeffective amount of OKT11 antibody to an individual in need of suchtreatment. By selective reaction with OKT11⁺ antigen, the effectiveamount of OKT11 antibody will reduce the excess of OKT11⁺ cells, thusameliorating the effects of the excess. Diagnostic and therapeuticcompositions comprising effective amounts of OKT11 antibody in admixturewith diagnostically or pharmaceutically acceptable carriers,respectively, are also included within the present invention.

Since peripheral T cells are responsible for graft rejection, oneexample of a therapeutic use of OKT11 antibody is the administration toa graft recipient of an amount of OKT11 antibody effective to reduce oreliminate the graft rejection. In this way OKT11 antibody could besubstituted for the anti-thymocyte globulin discussed above with asignificant increase in specificity.

What is claimed is:
 1. A monoclonal antibody of class IgG produced by ahybridoma formed by fusion of cells from a mouse myeloma line and spleencells from a mouse previously immunized with leukemic cells from a humanwith T-ALL, which antibody:(a) reacts with essentially all normal humanperipheral T cells and with approximately 95% of normal humanthymocytes, but not with normal human B cells or Null cells; (b) reactswith early, common, and mature human thymocytes and with inducer andcytotoxic/suppressor human T cells, but not with human prothymocytes;(c) defines a T cell population which is lower than normal levels inmyasthenia gravis and multiple sclerosis; higher than normal levels inacute graft versus host reaction, hyper IgE, acute infectiousmononucleosis, and primary biliary cirrhosis; and completely absent inall stages of Hodgkins disease and psoriasis.
 2. The monoclonal antibodyof claim 1 which is of subclass IgG₁.
 3. The monoclonal antibody ofclaim 1 which is produced from a hybridoma formed by fusion ofP3X63Ag8U1 myeloma cells and spleen cells from a CAF₁ mouse previouslyimmunized with human leukemic T-ALL cells.
 4. A monoclonal antibody ofclass IgG produced by a hybridoma formed by fusion of cells from a mousemyeloma line and spleen cells from a mouse previously immunized withhuman leukemic T-ALL cells which reacts with essentially all normalhuman peripheral T cells and with approximately 95 percent of normalhuman thymocytes, but not with normal human B cells or null cells. 5.Mouse monoclonal antibody which reacts with essentially all normal humanperipheral T cells and with approximately 95% of normal humanthymocytes, but not with normal human B cells or Null cells.
 6. Amonoclonal antibody which reacts with essentially all normal humanperipheral T cells and with approximately 95% of normal human thymocytesbut not with normal human B cells or Null cells prepared by the method,which comprises the steps of:(i) immunizing mice with human leukemicT-ALL cells; (ii) removing the spleens from said mice and making asuspension of the spleen cells; (iii) fusing said spleen cells withmouse myeloma cells in the presence of a fusion promoter; (iv) dilutingand culturing the fused cells in separate wells in a medium which willnot support the unfused myeloma cells; (v) evaluating the supernatant ineach well containing a hybridoma for the presence of antibody to Erosette positive purified T cells or human thymocytes; (vi) selectingand cloning a hybridoma producing antibody which reacts with essentiallyall normal human peripheral T cells and with approximately 95% of normalhuman thymocytes, but not with normal human B cells or Null cells; and(vii) recovering the antibody from the supernatant above said clones. 7.A monoclonal antibody which reacts with essentially all normal humanperipheral T cells and with approximately 95% of normal humanthymocytes, but not with normal human B cells or Null cells prepard bythe method, which comprises the steps of:(i) immunizing mice with humanleukemic T-ALL cells; (ii) removing the spleens from said mice andmaking a suspension of the spleen cells; (iii) fusing said spleen cellswith mouse myeloma cells in the presence of a fusion promoter; (iv)diluting and culturing the fused cells in separate wells in a mediumwhich will not support the unfused myeloma cells; (v) evaluating thesupernatant in each well containing a hybridoma for the presence ofantibody to E rosette positive purified T cells or human thymocytes;(vi) selecting and cloning a hybridoma producing antibody which reactswith essentially all normal human peripheral T cells and withapproximately 95% of normal human thymocytes, but not with normal humanB cells or Null cells; (vii) transferring said clones intraperitoneallyinto mice; and (viii) harvesting the malignant ascites or serum fromsaid mice, which ascites or serum contains the desired antibody.
 8. Amethod of preparing monoclonal antibody which reacts with essentiallyall normal human peripheral T cells and with approximately 95% of normalhuman thymocytes, but not with normal human B cells or Null cells, whichcomprises culturing the hybridoma ATCC CRL 8027 in a suitable medium andrecovering the antibody from the supernatant above said hybridoma. 9.The monoclonal antibody prepared by the method of claim
 8. 10. A methodof preparing monoclonal antibody which reacts with essentially allnormal human peripheral T cells and with approximately 95% of normalhuman thymocytes but not with normal human B cells or Null cells, whichcomprises injecting into a mouse the hybridoma ATCC CRL 8027 andrecovering the antibody from the malignant ascites or serum of saidmouse.
 11. The monoclonal antibody prepared by the method of claim 10.